Nerve growth factor (beta-NGF) is a neurotrophic factor playing a crucial role in the growth and survival of neurons (sensory and sympathetic) (Levi-Montalcini, R., Science 237 (1987) 1154; Thoenen, H., et al., Physiol. Rev. 60 (1980) 1284; Yankner, B. A., et al., Annu. Rev. Biochem. 51 (1982) 845). Beta-NGF belongs to a cysteine-knot superfamily of growth factors assuming stable dimeric protein structure. Furthermore, beta-NGF promotes the growth, differentiation and vitality of cholinergic neurons of the central nervous system (Hefti, F. J., J. Neurobiol. 25 (1994) 1418). Possible therapeutic indications for recombinant human nerve growth factor include peripheral sensory neuropathies, e.g. associated with diabetes or as a possible side effect in AIDS therapy. Other indications for beta-NGF are central neuropathies, e.g. Alzheimer's disease. In this case, the loss of memory is the result of a loss of cholinergic neurons. Beta-NGF has also been found to be effective in the treatment of human cutaneous and corneal ulcers (Bernabei et al. Lancet 1999; Lambiase et al. NEJM 1998). Moreover, Beta-NGF has also been shown to protect retinal cells from degeneration and apoptosis in an experimental animal model of glaucoma and to improve visual function in a few patients affected by glaucoma (Lambiase A, et al. PNAS 2009).
Mature human beta-NGF is a 118 amino acid protein which is translated as a preproprotein consisting of 241 amino acids. The signal peptide (prepeptide) of 18 amino acids is cleaved during translocation into the endoplasmic reticulum (ER). The resulting proprotein (proNGF) is processed at its N-terminus by removing the pro-sequence by protease cleavage. Mature human NGF shows a high degree of identity (about 90%) to rodent (murine and rat) beta-NGF. For clinical studies or therapeutic uses, beta-NGF has to be provided in high concentrations. Submaxillary glands of mice are a natural source of beta-NGF. However, these beta-NGF preparations are heterogeneous mixtures of different dimers and thus not suitable for therapeutic uses. Furthermore, it is desirable to administer the human form of the protein to patients. In human tissue, however, neurotrophic factors are present only in low concentrations.
The prosequence is a domain separate from the mature protein (see the sequence data in FIG. 1, wherein the prosequence is indicated in bold). These two domains are separated by an exposed protease cleavage site with a basic amino acid target sequence of the type Arg-Ser-Lys-Arg located at positions 101 to 104 of the human proNGF sequence (SEQ ID NO: 1). This motif is naturally a cleavage site for the serine endoprotease Furin. Additionally, the cleavage site may be specifically processed by other suitable proteases, preferably by proteases with substrate specificity of cleavage after the amino acid Arginine (Arg, R). For example, the protease trypsin cleaves after basic amino acids such as Lysine (Lys, K) or Arginine (Arg, R).
Methods for the preparation of biologically active beta-NGF from its inactive proform are well-known in the field of the art. For example, EP 0 994 188 B1 describes a method for the preparation of biologically active beta-NGF from its inactive pro-form having a poor solubility. According to this method, beta-NGF is obtainable from recombinant insoluble inactive proNGF which solubilized in a denaturing solution. Afterwards, the solubilized proNGF is transferred into a non- or weakly denaturing solution. The denatured proNGF assumes a biologically active conformation as determined by the disulfide bonds present in native beta-NGF. Subsequently, the prosequence of proNGF is cleaved off whereby active beta-NGF is obtained.
Human proNGF contains a native protease (Furin) cleavage site Arg-Ser-Lys-Arg, thus having the following sequence motif: R1SK3R4. For specific production processes such as those requiring “Good Manufacturing Practice” (GMP) quality levels, materials such as enzymes have to be provided in high quality. The protease Furin is currently not available as GMP-grade protease.
Therefore, an alternative protease, Trypsin (EC 3.4.21.4), was chosen to cleave proNGF to result in a mature beta-NGF protein. The serine protease Trypsin cleaves peptide chains at the carboxyl side of basic amino acids Arginine or Lysine. In human proNGF, the naturally occuring cleavage site in human proNGF contains three positions with basic amino acids (positions 101, 103, and 104 of SEQ ID NO: 1; alternatively referred to as R1, K3 and R4 herein). Thus, cleavage of proNGF by Trypsin may lead to numerous different cleaved products depending on where exactly cleavage occurs. Typical cleavage products are SK3R4-beta-NGF and R4-beta-NGF and mature beta-NGF. This problem is exacerbated since dimerization of the beta-NGF protein will lead to an even higher number (up to six) of inhomogenous products which have to be purified in following steps (see FIG. 2a).